Semipermanent p-nitrobenzyloxycarbonyl (pNZ) protection of Orn and Lys side chains: Prevention of undesired α-Fmoc removal and application to the synthesis of cyclic peptides

Article abstract of DOI:10.1016/j.tetlet.2005.09.043

Semipermanent side-chain protection of Orn and Lys with p-nitrobenzyloxycarbonyl (pNZ) for Fmoc/^tBu chemistry does not result in the unwanted removal of α-Fmoc that occurs when groups such as Alloc are used for the same application. Furthermore, pNZ can be used in conjuction with p-nitrobenzyl ester (pNB) to prepare cyclic peptides.

Full text of DOI:10.1016/j.tetlet.2005.09.043

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 7733–7736
Semipermanent p-nitrobenzyloxycarbonyl (pNZ) protection of
Orn and Lys side chains: prevention of undesired a-Fmoc
removal and application to the synthesis of cyclic peptides
a
a,b,
a,c,
*
´
*
Albert Isidro-Llobet, Mercedes Alvarez
and Fernando Albericio
aBarcelona Biomedical Research Institute, Barcelona Science Park, 08028 Barcelona, Spain
bLaboratory of Organic Chemistry, Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain
cDepartment of Organic Chemistry, University of Barcelona, 08028 Barcelona, Spain
Received 2 August 2005; revised 2 September 2005; accepted 7 September 2005
Available online 26 September 2005
Abstract—Semipermanent side-chain protection of Orn and Lys with p-nitrobenzyloxycarbonyl (pNZ) for Fmoc/tBu chemistry does
not result in the unwanted removal of a-Fmoc that occurs when groups such as Alloc are used for the same application. Further-
more, pNZ can be used in conjuction with p-nitrobenzyl ester (pNB) to prepare cyclic peptides.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
O
O
O2N
Solid phase has become the preferred mode for peptide
synthesis.1 The success of these syntheses depends to a
great extent on the coupling reagents used1,2 as well as
the temporary and permanent protecting strategies em-
ployed.1,3 Among the most useful temporary protection
groups for amino acid side chains is fluorenylmethoxy-
carbonyl (Fmoc), which is removed after the incorpora-
tion of each protected amino acid. In contrast, the tert-
butyl (tBu), which is removed at the end of the synthesis,
is the permanent group of choice for most peptides.4
Figure 1. Structure of the pNZ protecting group.
basic free amine that causes premature removal of Fmoc
groups.8 This side reaction can have disastrous effects
for a synthesis, cleaving up to 20% of Fmoc groups
present.
Herein reported is the use of p-nitrobenzyloxycarbonyl
(pNZ) as an alternative to Alloc for semipermanent
side-chain protection of Orn and Lys. The application
of pNZ to avoid undesired a-Fmoc removal and in the
synthesis of cyclic peptides is also discussed (Fig. 1).
In the case of cyclic5 or branched6 peptides, semiperma-
nent protecting groups must also be used. These groups
must be stable to the conditions used to remove the tem-
porary protecting group (i.e., piperidine for Fmoc) and
should be removed without affecting any permanent
protecting groups. Currently, the semipermanent pro-
tecting groups most widely used in tandem with the
Fmoc/tBu strategy are allyl derivatives.7 However, the
palladium complex used in their removal is expensive.
Furthermore, deprotection of allyloxycarbonyl (Alloc)
from the side chains of Orn and Lys generates a highly
2. Results and discussion
The pNZ group was first described by Carpenter and
Gish as an alternative to benzyloxycarbonyl (Z).9 It
has also been used for the protection of the e-amino
group of Lys.10 Very recently, the pNZ has been de-
scribed as a temporary protecting group for the a-func-
tionalities in solid-phase peptide synthesis.11 pNZ is
orthogonal to the most common solid-phase peptide
Keywords: Combinatorial chemistry; Orthogonal protecting group;
Side reactions.
t
*
synthesis protecting groups such as Bu/tert-butyloxy-
Corresponding authors. Tel.: +34 93 403 70 88; fax: +34 93 403 71
26; e-mail: albericio@pcb.ub.es
carbonyl (Boc), Fmoc, and Alloc, and is removed under
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.09.043

7734
A. Isidro-Llobet et al. / Tetrahedron Letters 46 (2005) 7733–7736
reductive and nearly neutral conditions with SnCl2 in
the presence of low concentrations of acid (1.6 mM
HCl/dioxane).11 Furthermore, its use circumvents typi-
cal side reactions associated with piperidine, such as
the formation of aspartiimides or diketopiperazines
(DKP).11
method similar to that used to avoid DKP formation
was used.11,14 If the a-Fmoc of Orn had been prema-
turely removed, then the byproduct {[H-Phe-Orn(&)-
Ala-NH2][H-Phe&]} (2) would have been generated, as
was observed upon following pathway b of Scheme 1.
As expected, peptide 2 was not detected by HPLC
(Fig. 2) in crude 1, indicating that premature Fmoc re-
moval had been avoided by using pNZ.
2.1. Avoiding undesired a-Fmoc removal
The basis for using pNZ to avoid DKP formation is that
its removal implies catalytic amounts of acid. Thus, the
deprotected amino acid is obtained as an ammonium
salt, leaving the basicity and nucleophilicity of the amine
function masked.11 The same principle was thus applied
to circumvent the unwanted removal of Fmoc caused by
the primary amines of Lys or Orn side chains upon
deprotection during solid-phase synthesis.
2.2. Use of pNZ/pNB protection for the solid-phase
synthesis of cyclic peptides
Similarly to the pair Alloc/Allyl used for the side-chain
protection of Lys or Orn and Glu or Asp, pNZ can be
used in conjunction with the related p-nitrobenzyl
(pNB) for the synthesis of side chain to side-chain cyclic
peptides.15 This was demonstrated for the synthesis of
the cyclic peptide H-Tyr-D-Ala-Phe-Glu(&)-Val-Val-
Lys(&)-NH2, a conformationally restricted analog of
deltorphin.16
To demonstrate the utility of pNZ as a temporary pro-
tecting group for the side chains of Orn and Lys, the
peptide {[H-Orn(&)-Ala-NH2][H-Phe&]}12 (1) was pre-
pared from Fmoc-Orn(pNZ)-OH (Scheme 1, pathway
a).13 After pNZ removal in mildly acidic conditions,
Fmoc-Phe-OH was directly coupled without a prior
neutralization step. Instead, an in situ neutralization
The peptide was synthesized on a Rink amide resin
using Fmoc-Lys(pNZ)-OH and Fmoc-Glu(OpNB)-
OH17 together with other Fmoc/tBu amino acids as
shown in Scheme 2. The elongation of the peptide chain
proceeded smoothly using N,N-diisopropylcarbodiimide
(DIPCDI)/hydroxybenzotriazole (HOBt) as coupling
agents. The semipermanent pNZ and pNB groups were
removed with SnCl2 and catalytic HCl. The cyclization
was then carried out using benzotriazol-1-yl-N-oxy-
H
Fmoc Orn Ala
pNZ
N
i) Fmoc removal
ii) pNZ removal
pNZ removal
H
tris(pyrrolidino)phosphonium
hexafluoro-phosphate
(PyBOP)/(N,N-diisopropylethyl amine (DIEA)). Final-
ly, cleavage of the peptidyl resin with trifluoroacetic acid
(TFA) rendered the target peptide with a yield of 14.1%
after HPLC purification (Fig. 3).
(a)
(b)
H
Fmoc Orn Ala
N
H Orn Ala
N
i) Fmoc-L-Phe-OH coupling
with "in situ" neutralization
ii) Fmoc removal
iii) TFA cleavage
3. Conclusions
H Orn Ala NH2
Phe
The use of pNZ as a temporary protecting group of
Orn and Lys avoids premature a-Fmoc removal after
side-chain deprotection of the x-amino function.
Furthermore, pNZ has been used in combination with
its derivative pNB to synthesize a cyclic peptide via
side-chain to side-chain cyclization. The pNZ/pNB
combination has thus been validated as a new member
H
Phe Orn Ala NH2
Phe
1
2
H
H
Scheme 1. Strategy used to demonstrate that premature a-Fmoc
elimination does not occur when pNZ is used to protect amino acid
side chains.
Figure 2. HPLC chromatograms of H-Orn(Phe)-Ala-NH2 (1) and H-Phe-Orn(Phe)-Ala-NH2 (2).

A. Isidro-Llobet et al. / Tetrahedron Letters 46 (2005) 7733–7736
7735
todiode Array). UV detection was performed at 220 nm,
and linear gradients of CH3CN (0.036% TFA) into H2O
(0.045% TFA) were run at a flow rate of 1.0 mL/min
(Gradient A: from H2O to CH3CN in 15 min; Gradient
B: from CH3CN–H2O (80:20) CH3CN–H2O (65:35) in
15 min).
H2N
Rink amide resin
i) Fmoc-L-Lys(pNZ) (4 eq), HOBt (4 eq),
DIPCDI (4 eq)
ii) capping: Ac2O (10 eq), DIEA (5 eq)
H
N
Fmoc-Lys(pNZ)
4.1. Avoiding undesired a-Fmoc removal
coupling of the remaining amino acids
To a Rink amide resin (f = 0.66 mmol/g, 100 mg),
placed in a 2 mL polypropylene syringe fitted with a
polyethylene filter disk, was added Fmoc-Ala-OH
(4 equiv) in the presence of equimolar amounts of DIP-
CDI and HOBt in N,N-dimethylformamide (DMF).
The mixture was stirred for 1 h, at which point the Fmoc
group was removed with piperidine–DMF (1:4). Fmoc-
Orn(pNZ)-OH (4 equiv) was then coupled as described
above. The resin was dissolved in DMF and divided into
two equal portions (a,b).
Fmoc-Tyr(tBu)-D-Ala-Phe-Glu(OpNB)-Val-Val-Lys(pNZ) NH
pNZ and pNB removals: 6M SnCl2,
1.6 mM HCl−dioxane in DMF (2 x 45 min)
Fmoc-Tyr(tBu)-D-Ala-Phe-Glu-Val-Val-Lys NH
Cyclization: PyBOP (4 eq), DIEA (12 eq),
2 x 24 h.
Fmoc-Tyr(tBu)-D-Ala-Phe-Glu-Val-Val-Lys NH
The pNZ group of resin a was removed using 6 M SnCl2
and 1.6 mM HCl/dioxane in DMF (2 · 30 min), and
Fmoc-Phe-OH was subsequently coupled with PyBOP
(5 equiv) and DIEA (8 equiv) in DMF. The Fmoc
groups were removed, the peptidyl resin was cleaved
with TFA/DCM/H2O (90:5:5), and crude 1 was concen-
trated by evaporation, then characterized by HPLC
(tR = 5.39 min, gradient A) and ESMS (calcd for
C26H36N6O4, 496.3, found m/z, 497.5 [M+H]+).
NH
O
Fmoc removal: piperidine−DMF (2:8)
Cleavage: TFA−H2O−DCM (90:5:5)
H-Tyr-D-Ala-Phe-Glu-Val-Val-Lys NH2
NH
O
Scheme 2. Solid-phase synthesis of the deltorphin cyclic analog,
H-Tyr-D-Ala-Phe-Glu(&)-Val-Val-Lys(&)-NH2.
The Fmoc and pNZ groups of b were removed as previ-
ously described and Fmoc-Phe-OH was coupled as
above. Then, Fmoc group was removed and the peptide
submitted to the same protocol as above to give 2, which
was dissolved in CH3CN–H2O and characterized by
HPLC (tR = 4.42 min, gradient A) and ESMS (calcd
for C17H27N5O3, 349.2, found m/z, 350.4 [M+H]+).
4.2. Preparation of the cyclic deltorphin analog H-Tyr-D-
Ala-Phe-Glu(&)-Val-Val-Lys(&)-NH2
Fmoc-Lys(pNZ)-OH (0.9 equiv), HOBt (0.9 equiv), and
DIPCDI (0.9 equiv) in DMF were added to Rink amide
resin (0.2 g, f = 0.68 mmol/g) and stirred for 1 h. The
resin was then treated with Ac2O (10 equiv) and DIEA
(5 equiv) in DMF for 30 min in order to acetylate the
free amino groups. The new loading of the resin was
calculated by UV titration of the Fmoc group
(f = 0.49 mmol/g). The remaining amino acids were
coupled using 4 equiv each of Fmoc-Aaa-OH/DIP-
CDI/HOBt in DMF. Once the linear peptide was assem-
bled, the Fmoc group of the last residue (Tyr) was left
on, and the pNZ and pNB groups were removed by
treating the resin with 6 M SnCl2 and 1.6 mM HCl/diox-
ane in DMF (2 · 45 min). An aliquot of the resin was
then treated with piperidine/DMF (1:4) to remove the
Fmoc group, treated with TFA/H2O/DCM (90:5:5) to
cleave the peptide from the resin, and analyzed by
HPLC (tR = 6.08 min, gradient A) and ESMS (calcd
for C42H63N9O10, 853.5, found m/z, 854.2 [M+H]+).
Figure 3. HPLC chromatogram of the cyclic deltorphin analog H-Tyr-
D-Ala-Phe-Glu(&)-Val-Val-Lys(&)-NH2.
of the family of orthogonal protecting group pairs18
used in the synthesis of cyclic peptides as well as other
complex peptides and organic molecules. Using the
pNZ/pNB pair in conjunction with the Alloc/Allyl pair
could enable regioselective synthesis of bicyclic peptides.
4. Experimental section
Analytical HPLC was carried out in a Waters instru-
ment comprising two solvent-delivery pumps and auto-
matic injector (Waters Separations Module 2695) and
a variable-wavelength detector (model Waters 996 Pho-
The remaining resin was treated with PyBOP (4 equiv)
and DIEA (12 equiv) in DMF for 24 h to cyclize the

7736
A. Isidro-Llobet et al. / Tetrahedron Letters 46 (2005) 7733–7736
Barany, G. In Synthetic Peptides: A UserÕs Guide, 2nd ed.;
Grant, G. A., Ed.; W. H. Freeman & Co.: New York,
2001; pp 93–219.
peptide. An aliquot of the resin was then treated with
piperidine/DMF (1:4), TFA/H2O/DCM (90:5:5), and
analyzed by HPLC and HPLC–MS, which indicated
that the crude product comprised 65% linear peptide
and 35% cyclic peptide. The remaining resin was re-
treated with the same reagents and after 24 h an aliquot
of the resin was analyzed as described above, indicating
50% linear peptide and 50% cyclic peptide. The cycliza-
tion was completed by stirring the resin with PyBOP
(4 equiv), DIEA (12 equiv), and HOAt (8 equiv) in
DMF for 72 h. The Fmoc group was then removed
and the peptide was cleaved from the resin with TFA/
H2O/DCM (90:5:5). The TFA was removed by evapora-
tion and the crude was dissolved in CH3CN/H2O (7:3)
and washed three times with CHCl3. H2O was added
to the aqueous layer, which was lyophilized and found
to contain a 1:1 mixture of the linear and cyclic peptides
among other impurities. The crude product was purified
by semipreparative HPLC to afford 1.8 mg of the cyclic
peptide, which was characterized by HPLC (tR =
6.26 min, 99% of purity, gradient B) and ESMS (calcd
for C42H63N9O10, 835.5, found m/z, 836.7 [M+H]+).
5. Rovero, P. In Solid-Phase Synthesis. A Practical Guide;
Kates, S. A., Albericio, F., Eds.; Marcel Dekker: New
York, NY, USA, 2000; pp 331–364.
6. Kates, S. A.; Daniels, S. B.; Albericio, F. Anal. Biochem.
1993, 212, 303–310.
´
7. Guibe, F. Tetrahedron 1997, 53, 13509–13556, and 1998,
54, 2967–3042.
8. (a) Farrera-Sinfreu, J.; Royo, M.; Albericio, F. Tetra-
hedron Lett. 2002, 43, 7813–7815; (b) Vig, B. S.; Murray,
T. F.; Aldrich, J. V. Biopolymers 2003, 71, 620–637.
9. (a) Carpenter, F. H.; Gish, D. T. J. Am. Chem. Soc. 1952,
74, 3818–3821; (b) Gish, D. T.; Carpenter, F. H. J. Am.
Chem. Soc. 1953, 75, 950–952; (c) Shields, J. E.; Carpenter,
F. H. J. Am. Chem. Soc. 1961, 83, 3066–3070.
10. (a) Hocker, M. D.; Caldwell, C. G.; Macsata, R. W.;
Lyttle, M. H. Pept. Res. 1995, 8, 310–315; (b) Peluso, S.;
Dumy, P.; Nkubana, C.; Yokokawa, Y.; Mutter, M. J.
Org. Chem. 1999, 64, 7114–7120.
´
11. Isidro-Llobet, A.; Guasch-Camell, J.; Alvarez, M.; Alberi-
cio, F. Eur. J. Org. Chem. 2005, 14, 3031–3039.
12. The nomenclature used for branched and cyclic peptides is
´
described in: Spengler, J.; Jimenez, J. C.; Giralt, E.;
Albericio, F. J. Pept. Res. 2005, 65, 550–555.
13. Fmoc-Orn(pNZ)-OH and Fmoc-Lys(pNZ)-OH were syn-
thesized from Fmoc-Aaa(Boc)-OH and p-nitrobenzyl
chloroformate as starting materials, by the azide method:
Cruz, L. J.; Beteta, N. G.; Ewenson, A.; Albericio, F. Org.
Proc. Res. Dev. 2004, 8, 920–924.
14. (a) Gairi, M.; Lloyd-Williams, P.; Albericio, F.; Giralt, E.
Tetrahedron Lett. 1990, 31, 7363–7366; (b) Alsina, J.;
Giralt, E.; Albericio, F. Tetrahedron Lett. 1996, 37, 4195–
4198.
Acknowledgments
This work was partially supported by CICYT (BQU
2003-00089, PETRI 95-0658-OP), the Generalitat de
Catalunya (Grup Consolidat and Centre de Referencia
en Biotecnologia), and the Barcelona Science Park.
A.I. thanks the Generalitat de Catalunya for a predoc-
toral fellowship.
`
15. The pNB group has been used to protect the C-terminal
carboxylic group in side-chain or backbone anchoring
strategies for the solid-phase synthesis of head-to-tail
cyclic peptides. Romanovskis, P.; Spatola, A. F. J. Pept.
Res. 1998, 52, 356–374; Royo, M.; Farrera-Sinfreu, J.;
References and notes
´
1. (a) Stewart, J. M.; Young, J. D. Solid-Phase Peptide
Synthesis, 2nd ed.; Pierce Chemical Co.: Rockford, IL,
1984; (b) Lloyd-Williams, P.; Albericio, F.; Giralt, E.
Chemical Approaches to the Synthesis of Peptides and
Proteins; CRC: Boca Raton, FL, USA, 1997; (c) Houben-
Weyl, Synthesis of Peptides and Peptidomimetics; Good-
man, M., Felix, A., Moroder, L. A., Toniolo, C., Eds.;
Georg Thieme Verlag: Stuttgart, Germany, 2002; Vol. E
22a-e; (d) Bruckdorfer, T.; Marder, O.; Albericio, F. Curr.
Pharm. Biotechnol. 2004, 5, 29–43.
Sole, L.; Albericio, F. Tetrahedron Lett. 2002, 43, 2029–
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16. Schiller, P. W.; Weltrowska, G.; Nguyen, T. M.-D.;
Wilkes, B. C.; Chung, N. N.; Lemieux, C. J. Med. Chem.
1992, 35, 3956–3961.
17. Synthesis of Fmoc-L-Glu(O pNB)-OH: Fmoc-L-Glu-OtBu
(3.25 g, 7.63 mmol, 1.1 equiv) and p-nitrobenzyl alcohol
(1.06 g, 6.94 mmol, 1 equiv) were dissolved in a minimum
amount of DCM (7.5 mL). DIPCDI (2.16 g, 6.94 mmol,
1 equiv) and DMAP (0.085 g, 0.69 mmol, 0.1 mmol) were
added. The reaction was followed by TLC (DCM) and
after 2 h, EtOAc (40 mL) was added, and the solution was
washed with 10% aqueous Na2CO3 (3 · 25 mL). The
organic layer was dried over MgSO4 and the solvent was
removed under vacuum to afford a yellow oil (3.42 g, 88%
yield).
2. (a) Kates, S. A.; Albericio, F. In Solid-Phase Synthesis. A
Practical Guide; Kates, S. A., Albericio, F., Eds.; Marcel
Dekker: New York, NY, USA, 2000; pp 275–330; (b)
´
Albericio, F.; Chinchilla, R.; Dodsworth, D.; Najera, C.
Org. Prep. Proc. Int. 2001, 33, 203–303.
3. (a) Wade, J. D. In Solid-Phase Synthesis. A Practical
Guide; Kates, S. A., Albericio, F., Eds.; Marcel Dekker:
New York, NY, USA, 2000; pp 103–128; (b) Doherty-
Kirby, A. L.; Lajoie, G. In Solid-Phase Synthesis. A
Practical Guide; Kates, S. A., Albericio, F., Eds.; Marcel
Dekker: New York, NY, USA, 2000; pp 129–195.
4. (a) Fmoc Solid Phase Peptide Synthesis; Chan, W. C.,
White, P. D., Eds.; Oxford University Press: Oxford (UK),
2000; (b) Fields, G. B.; Lauer-Fields, J. L.; Liu, R.-q.;
Fmoc-L-Glu(ONB)-OtBu (1.68 g, 3 mmol) was dissolved
in TFA/DCM (1:1) (20 mL) and stirred for 1 h. The
solvent was then eliminated in vacuo and diethyl ether was
added and evaporated four times in order to ensure
that the TFA was removed. After drying the product in
vacuo, a white solid was obtained (1.51 g, 3 mmol, nearly
quantitative yield).
18. Albericio, F. Biopolym. (Pept. Sci.) 2000, 55, 123–139.